Ultra‐acceleration of Photochemical Cytosine Deamination by Using a 5′‐Phosphate‐Substituted Oligodeoxyribonucleotide Probe Containing a 3‐Cyanovinylcarbazole Nucleotide at Its 5′‐End

• Published in: Chembiochem : a European journal of chemical biology Vol. 19; no. 21; pp. 2257 - 2261
• Main Authors: Sethi, Siddhant, Honda, Nozomi, Wan, Licheng, Nakamura, Shigetaka, Fujimoto, Kenzo
• Format: Journal Article
• Language: English
• Published: Germany Wiley Subscription Services, Inc 02.11.2018
• Subjects: Backbone; Cytosine; cytosine deamination; Deamination; Disorders; DNA; DNA/RNA editing; Genes; High temperature; Hydrogen bonding; Mutation; Oligonucleotides; Phosphates; photo-crosslinking; Photochemicals; Proteins; RNA; Site-directed mutagenesis; Temperature requirements; Water chemistry; DNA/RNA editing; photo-crosslinking; RNA; DNA; cytosine deamination
• ISSN: 1439-4227; 1439-7633
• DOI: 10.1002/cbic.201800384

Genes are the blueprints for the architectures of living organisms, providing the backbone of the information required for formation of proteins. Changes in genes lead to disorders, and these disorders could be rectified by reversing the mutations that caused them. Photochemical methods currently in use for site‐directed mutagenesis employ the photoactive 3‐cyanovinylcarbazole (CNVK) nucleotide incorporated in the oligodeoxyribonucleotide (ODN) backbone. The major drawback of this method, the requirement for high temperature, has been addressed, and deamination has previously been achieved at 37 °C but with low efficiency. Here, efficient deamination has been accomplished under physiological conditions by using a short complementary photoactive ODN with a 5′‐phosphate group in the −1 position with respect to the target cytosine. It is hypothesized that the free phosphate group affects the microenvironment around the target cytosine by activating the incoming nucleophile through hydrogen bonding with the water molecule, thus facilitating nucleophilic attack on the cytosine C‐4 carbon. The degree of deamination observed in this technique is high and the effect of the phosphate group is to accelerate the deamination reaction. Targeted deamination of a cytosine base in a short ODN to afford a uracil system under physiological conditions was achieved by using a 5′‐phosphate group in the −1 position (with respect to the target cytosine) in a complementary photoactive ODN. It is hypothesized that the free phosphate group affects the microenvironment around the target cytosine unit.